Osteoporosis is responsible for about 1.5 million fractures each year in the United States, of which about 300,000 are hip fractures. Fifty to 75% of patients with hip fractures are unable to live independently, resulting in increased costs of care. Osteoporosis is characterized by a greater than normal loss of bone density as people age. This disease occurs with a high frequency (>30% of females over age 60) in Western and Asian cultures, and is increasing in prevalence as longevity increases. While the exact cause of these bone repair disorders is unknown, it is clear the dynamic process of bone remodeling is disrupted in a process characterized by a decrease in osteoblastic (bone-producing cells) activity and an increase in osteoblastic (bone degrading cells) activity (Parfitt (1992) Triangle 31:99-110; Parfitt (1992) In Bone, volume 1, B. K. Hall, ed. Teleford Press and CRC Press, Boca Raton, Fla., p. 351-429).
The use of bone grafts is conventional practice in orthopedics, neurosurgery and dentistry, as well as in plastic/reconstruction surgery and this utilization has been growing in frequency over the past two decades. With the exception of blood, bone is the most frequently transplanted tissue with an estimated 500,000 bone grafts used in the US annually. Common orthopedic uses of bone grafts include the management of non-unions and acute long bone fracture, joint reconstruction and to facilitate fusion of vertebral motion segments in treating a variety of spinal disorders (Lane (1987) Ortho Clin N Amer 18:213-225).
Currently, the most clinically acceptable grafting material is autologous bone. So-called autografts are often obtained from a secondary operative site. There are significant issues associated with autografts. These include lack of an adequate supply for large wounds or defects. Elderly individuals with osteoporosis or osteopenia make the use of an autograft problematic. The secondary morbidity associated with the harvesting operation is high. These complications include infections, pelvic instability (the bone is often harvested from the iliac crest), hematoma, and pelvic fracture (Laurie et al. (1984) Plas Rec Surg 73:933-938; Summers et al. (1989) J Bone Joint Surg 71B:677-680; Younger et al. (1989) J Orthop Trauma. 3:192-195; Kurz et al. (1989) Spine 14:1324-1331). In addition, chronic pain at the donor site is the second most frequently reported complication (Turner et al. (1992) JAMA 268:907-911). Finally, the ability to shape the autograft to the defect/wound site is limited due to the rigid nature of the material.
Recent investigations have focused on the use of a variety of matrices, either inorganic such as hydroxyapatite (Flatley et al. (1983) Clin Orthop Rel Res 179:246-252; Shima et al. (1979) J Neurosurg 51:533-538; Whitehill et al. (1985) Spine 10:32-41; Herron, et al. (1989) Spine 14:496-500; Cook et al. (1986) Spine 11:305-309; the contents of which are incorporated herein by reference) or organic such as demineralized bone matrix (DBM) (reviewed in Ashay et al. (1995) Am J Orthop 24:752-761; the contents of which are incorporated herein by reference). These matrices are thought to be osteoconductive (facilitate the invasion of bone forming cells in an inert matrix) or osteoinductive (induce the transformation of recruited precursor cells to osteoblasts). A number of successful clinical outcomes have been observed with some of these products approved for use clinically by the Food and Drug Administration. In spite of these successes, a number of issues remain for the utility of these matrices. The first is the variable subject response to DBM. Also these matrices take much longer than autologous bone transplantation to develop significant structural integrity and bear load effectively.
An alternative to transplantation and the use of simple matrices is the admixture of bone marrow or bone marrow stromal cells with DBM. Ideally the cells and DBM will be derived from the same subject although allogeneic DBM has already been used clinically with initial success (Mulliken et al. (1981) Ann Surg 194:366-372; Kaban et al. (1982) J Oral Maxillofac Surg 40:623-626). Transplantation methods using autologous bone marrow cells with allogeneic DBM have yielded good results (Connolly (1995) Clin Orthop 313:8-18). However, issues that may impact the widespread use of these techniques include potential for contamination by non-self materials, the acceptability of the patient for donating bone marrow, and the potential complications that arise from bone marrow aspirations and depletion of bone marrow from the source.
A number of groups have shown that bone marrow stromal cells and cell lines derived thereof are capable of differentiating into cells biochemically and morphologically similar to osteoblasts (Dorheim et al. (1993) J Cell Physiol 154:317-328; Grigoriadis et al. (1988) J Cell Biol 106:2139-2151; Benayahu et al. (1991) Calcif Tiss Int. 49:202-207; the contents of which are incorporated by reference). In most cases, fibroblast-like cells were isolated from human or animal bone marrow and plated onto standard tissue cultureware. Generally, a standard media formulation, such as Dulbecco's Modified Eagle's Medium (DMEM) plus fetal calf serum 10-20% and antibiotics is used to select for the enrichment of these cells (Ashton et al. (1980) Clin Orthop 151:294-307; Sonis et al. (1983) J Oral Med 3:117-120). Cells were then stimulated to differentiate into osteoblasts by changing the medium to one containing 5-20% fetal calf serum, 2-20 mM β-glycerophosphate and 20-75 μM ascorbic acid or ascorbic-2-phosphate (Asahina et al. (1996) Exp Cell Res 222:38-47; Yamaguchi et al. (1991) Calcif Tissue Int 49:221-225; the contents of which are incorporated herein by reference). After 14-21 days in culture, many of these cell types and cell lines will mineralized matrices on the cultureware as evidenced by positive von Kossa staining. Other phenotypic indicators of osteoblast lineage include elevated secreted alkaline phosphatase activity; the presence of secreted osteocalcin in the media; and the increased expression-of several genes thought to be specifically expressed in osteoblasts, including osteocalcin, osteopontin, and bone sialoprotein (Stein et al. (1990) FASEB J 4:3111-3123; Dorheim et al. (1993) J Cell Physiol 154:317-328; Asahina et al. (1996) Exp Cell Res 222:38-47; Yamaguchi et al. (1991) Calcif Tissue Int 49:221-225).
There have been a number of detailed studies carried out in several laboratories demonstrating that transplanted bone marrow stromal cells can form ectopic bone (Gundle et al. (1995) Bone 16:597-603; Haynesworth et al. (1992) Bone 13:81-89; Boynton et al. (1996) Bone 18:321-329). For example, human and murine bone marrow stromal fibroblasts have been transplanted into immunodeficient SCID mice (Krebsbach et al. (1997) Transplantation 63:1059-1069; Kuznetsov et al. (1997) J Bone Min Res 12:1335-1347). Using antibody and histochemical markers, it was demonstrated that the donor bone marrow stromal cells account for the newly developed osteoblasts at sites of ectopic bone formation in the presence of an inductive matrix. Murine cells formed bone in the presence of hydroxyapatite/tricalcium phosphate particles (HAITCP), gelatin, poly-L-lysine, and collagen. In contrast, human stromal cells efficiently formed bone only in the presence HA/TCP. No exogenous BMP was required in these studies.
Bone formation is not limited to the skeleton. For example, the introduction of ceramic or demineralized bone matrix into intramuscular, subrenal capsular or subcutaneous sites will result in bone formation if the area is simultaneously expressing bone morphogenetic protein (Urist (1965) Science 150:893.-899). These results suggest that cells present in these tissues have some capability of forming bone precursor cells under the proper environmental conditions.
Ectopic bone formation in soft tissue such as fat is a rare pathologic condition observed in patients with fibrosis ossificans progressiva, an inherited disease. While the etiology of the disease is not completely understood, it arises in part from the abnormal expression of BMP by lymphocytes localized to sites of soft tissue injury (Kaplan et al. (1997) J Bone Min Res 12:855; Shafritz et al. (1996) N Engl J Med 335:555-561]). Bone formation is also observed on rare occasions in lipomas (Katzer (1989) Path Res Pract 184:437-443).
The stromal-vascular fraction isolated from adipose tissue after collagenase treatment has been demonstrated to contain a large quantity of preadipocytes, or cells that are predisposed to differentiate into adipocytes (Hauner et al. (1989) J Clin Invest 34:1663-1670). These cells can spontaneously differentiate into adipocyies at relatively low frequency or respond to adipogenic agonists such as thiazolidinediones to a much higher frequency of differentiation (Halvorsen (1997) Strategies 11:58-60; Digby (1997) Diabetes 4:138-141). There is evidence to suggest that stromal cells exhibit a reciprocal pattern of differentiation between these lineages (Gimble et al. (1996) Bone 19:421-428; Bennett et al. (1991) J Cell Sci 99:131-139; Beresford et al. (1992) J Cell Sci 102:341-351). Specifically, adipogenesis is accompanied by reduced osteoblastic potential while osteogenesis is accompanied by reduced adipogenic potential.
Under certain conditions, bone marrow stromal cells can be differentiated into adipocytes. In fact, several bone stromal cell lines have been extensively characterized with respect to this ability (Gimble et al. (1990) Eur J Immunol 20:379-387; Gimble et al. (1992) J Cell Biochem 50:73-82; Gimble et al. (1996) Bone 19:421-428). However, prior to the instant invention, it was not known that stromal cells isolated from adipose tissue could be made to differentiate into osteoblasts.